Molecular phylogeny of Vincetoxicum (Apocynaceae, Asclepiadoideae) from Thailand and integrative taxonomy corroborating a new cryptic species within Vincetoxicum kerrii.

An updated phylogeny of the genus Vincetoxicum s.l. based on DNA sequences of the nuclear internal transcribed spacer (ITS) region and three plastid markers is presented. In total, 21 accessions newly sequenced from Thailand were added to the dataset of the homologous sequences of 75 other Vincetoxicum taxa downloaded from GenBank. In our analysis, the relationships between the well-supported clades largely correspond to those revealed in previous studies. With some exceptions, the phylogenetic positions of the Thai taxa in relation to other conspecifics and congeners generally reflect the geographic distributions of taxa. Moreover, recent extensive sampling throughout Thailand and in-depth investigation have revealed V. kerrii, a slender twiner widespread from South China to Indo-China, to be a species complex. A combination of molecular, morphological, anatomical, ultrastructural and ecological evidence allowed us to reveal a new cryptic species hidden within V. kerrii, described here under the name V. simplex. A comprehensive description, illustrations, photographs, and comparison with the morphologically similar species are provided. Although V. simplex and V. kerrii s.s. resemble one another in various aspects of vegetative and reproductive structures, the latter is phylogenetically closely related to V. irrawadense, which is much less similar morphologically to both V. simplex and V. kerrii s.s. than the latter two are to each other. In addition to the new cryptic species recognized in the present study, a new record for Thailand, V. microstachys, is also reported.

Evolution of 101 Apocynaceae plastomes and phylogenetic implications.

Apocynaceae are one of the ten species-richest angiosperm families. However, the backbone phylogeny of the family is yet less well supported, and the evolution of plastome structure has not been thoroughly studied for the whole family. Herein, a total of 101 complete plastomes including 35 newly sequenced, 24 reassembled from public raw data and the rest from the NCBI GenBank database, representing 26 of 27 tribes of Apocynaceae, were used for comparative plastome analysis. Phylogenetic analyses were conducted using a combined plastid data matrix of 77 protein-coding genes from 162 taxa, encompassing all tribes and 41 of 49 subtribes of Apocynaceae. Plastome lengths ranged from 150,897 bp in Apocynum venetum to 178,616 bp in Hoya exilis. Six types of boundaries between the inverted repeat (IR) regions and single copy (SC) regions were identified. Different sizes of IR expansion were found in three lineages, including Alyxieae, Ceropegieae and Marsdenieae, suggesting multiple expansion events of the IRs over the SC regions in Apocynaceae. The IR regions of Marsdenieae evolved in two ways: expansion towards the large single copy (LSC) region in Lygisma + Stephanotis + Ruehssia + Gymnema (Cosmopolitan clade), and expansion towards both LSC and small single copy (SSC) region in Dischidia-Hoya alliance and Marsdenia (Asia-Pacific clade). Six coding genes and five non-coding regions were identified as highly variable, including accD, ccsA-ndhD, clpP, matK, ndhF, ndhG-ndhI, trnG(GCC)-trnfM(CAU), trnH(GUG)-psbA, trnY(GUA)-trnE(UUC), ycf1, and ycf2. Maximum likelihood and Bayesian phylogenetic analyses resulted in nearly identical tree topologies and produced a well-resolved backbone comprising 15 consecutive dichotomies that subdivided Apocynaceae into 15 clades. The subfamily Periplocoideae were embedded in the Apocynoid grade and were sister to the Echiteae-Odontadenieae-Mesechiteae clade with high support values. Three tribes (Melodineae, Vinceae, and Willughbeieae), the subtribe Amphineuriinae, and four genera (Beaumontia, Ceropegia, Hoya, and Stephanotis) were not resolved as monophyletic. Our work sheds light on the backbone phylogenetic relationships in the family Apocynaceae and offers insights into the evolution of Apocynaceae plastomes using the most densely sampled plastome dataset to date.

Multiple origins of two Ochrosia (Apocynaceae) species endemic to the Bonin (Ogasawara) Islands.

The genus, Ochrosia, is widely distributed from the West Indian Ocean throughout tropical Asia to the Middle Southern Pacific region. Ochrosia comprises many island-endemic species, suggesting that long-distance dispersal and isolation after migration are key factors for clarifying the diversification process. However, the phylogeny and biogeography of endemic Ochrosia species have not been evaluated well due to the difficulty of adequate sampling from the entire distribution range of the genus. In this study, we focused on two Ochrosia species endemic to the Bonin (Ogasawara) Islands in the northwest Pacific. The Bonin Islands are of volcanic origins and consist of two islands groups, the Ogasawara and Volcano Islands groups, approximately 300 km apart. Ochrosia nakaiana is endemic to the Ogasawara Islands group, whereas O. hexandra is endemic to the Volcano Islands group. To elucidate the phylogenetic positions of these two endemic Ochrosia species, we conducted molecular phylogenetic studies with dating and biogeographic analyses including other Ochrosia species. The phylogenetic trees showed that the two endemic species had distinct origins; O. nakaiana was closely related to O. oppositifolia and O. iwasakiana, whereas O. hexandra was related to O. mariannensis. Based on the chloroplast DNA phylogeny, the genus, Ochrosia, divided into two major lineages 36.6 million years ago. Further, the two endemic species of the Bonin Islands were independently derived approximately 1-2 million years ago. Ochrosia nakaiana originated from the Southeast Asia, New Caledonia, or other Pacific Islands, while O. hexandra derived from O. mariannensis in Micronesia. We demonstrated different origins of the two endemic Ochrosia species on the Bonin Islands. This study provided an excellent example of the complex origins and speciation of flora in the oceanic islands.

Population Genetics Coupled Chemical Profiling for Conservation Implications of Decalepis salicifolia (Bedd. ex Hook.f.) Venter, an Endemic and Critically Endangered Species of Western Ghats, India.

Information on the genetic diversity and population structure is essential for developing conservational management programs, especially for threatened species. Decalepis salicifolia (Bedd. ex Hook.f.) Venter is a steno-endemic and critically endangered species of the south Western Ghats of India. The present study used ISSR markers as well as essential oil profiling to reveal the extent and distribution of genetic as well as the chemical diversity of all the twelve known populations of D. salicifolia. A total of 84 amplicons generated using 17 ISSR primers represented an overall 72.34% polymorphism. The highest percentage of polymorphic loci was recorded in the population of Theemalai (40.48%) and lowest in Kokanmalai (4.76%) with an average of 20.04% across all the studied populations. At the species level, the Nei's genetic diversity observed was 0.255 ± 0.186, while Shannon's information index observed was 0.385 ± 0.260. The genetic similarity-based unweighted pair-group method with arithmetic average dendrogram grouped the populations according to their geographic locations, which was corroborated by principal component analysis and Bayesian clustering. Distribution of genetic variance through analysis of molecular variance indicated that 38% variance resides within the population, and 62% variance resides among the populations (P < 0.001). Gas chromatography analyses of root volatiles showed significant variation in the percent content of 2-hydroxy-4-methoxybenzaldehyde. The Mantel test analyses showed a positive correlation between the genetic versus geographic distances. Based on the results, both ex situ and in situ conservation strategies are suggested to maximally preserve the genetic resources of this endangered species.

High-throughput metabolomic and transcriptomic analyses vet the potential route of cerpegin biosynthesis in two varieties of Ceropegia bulbosa Roxb.

MAIN CONCLUSION: Exploration with high-throughput transcriptomics and metabolomics of two varieties of Ceropegia bulbosa identifies candidate genes, crucial metabolites and a potential cerpegin biosynthetic pathway. Ceropegia bulbosa is an important medicinal plant, used in the treatment of various ailments including diarrhea, dysentery, and syphilis. This is primarily attributed to the presence of pharmaceutically active secondary metabolites, especially cerpegin. As this plant belongs to an endemic threatened category, genomic resources are not available hampering exploration on the molecular basis of cerpegin accumulation till now. Therefore, we undertook high-throughput metabolomic and transcriptomic analyses using different tissues from two varieties namely, C. bulbosa var. bulbosa and C. bulbosa var. lushii. Metabolomic analysis revealed spatial and differential accumulation of various metabolites. We chemically synthesized and characterized the cerpegin and its derivatives by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Importantly, these comparisons suggested the presence of cerpegin and 5-allyl cerpegin in all C. bulbosa tissues. Further, de novo transcriptome analysis indicated the presence of significant transcripts for secondary metabolic pathways through the Kyoto encyclopedia of genes and genomes database. Tissue-specific profiling of transcripts and metabolites showed a significant correlation, suggesting the intricate mechanism of cerpegin biosynthesis. The expression of potential candidate genes from the proposed cerpegin biosynthetic pathway was further validated by qRT-PCR and NanoString nCounter. Overall, our findings propose a potential route of cerpegin biosynthesis. Identified transcripts and metabolites have built a foundation as new molecular resources that could facilitate future research on biosynthesis, regulation, and engineering of cerpegin or other important metabolites in such non-model plants.

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study.

Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

Testing the hypothesis of loss of defenses on islands across a wide latitudinal gradient of Periploca laevigata populations.

PREMISE OF THE STUDY: We tested a hypothesis that predicts loss of chemical defenses on island plant populations (LCDIH) as an evolutionary response to limited herbivore pressures. METHODS: Using a common garden approach, we grew 16 populations (N = 286 seedlings) of Periploca laevigata, a Mediterranean shrub for which previous studies suggested that animal browsing elicits defensive responses mediated by tannins. Our experimental setting represented a wide latitudinal gradient (37-15°N) encompassing three island systems, virtually free of large herbivores, and three mainland areas. Putative chemical defenses were estimated from tannin-protein precipitation assays, and inducible responses in growth and chemical traits were assessed between seasons and by subjecting plants to a pruning treatment. KEY RESULTS: We failed to find support for the LCDIH, since island populations (Canary Islands, Cape Verde) had increasingly higher constitutive levels of tannins at lower latitudes. Seasonality, but not experimental pruning, induced variation in levels of tannins in a consistent pattern across populations. Thus, net differences in leaf tannin concentration remained similar among geographical areas regardless of the factor considered, with latitude being the best explanatory factor for this trait over seasonal growth patterns. CONCLUSIONS: Geographical variation in total tannin pools appears to be mediated by factors other than herbivore pressure in P. laevigata. We hypothesize that abiotic correlates of latitude not considered in our study have promoted high constitutive levels of leaf tannins across Macaronesian populations, which ultimately may explain the pattern of seasonal variation and latitudinal increase from Mediterranean to subtropical Cape Verde populations.

Resolution effects in reconstructing ancestral genomes.

BACKGROUND: The reconstruction of ancestral genomes must deal with the problem of resolution, necessarily involving a trade-off between trying to identify genomic details and being overwhelmed by noise at higher resolutions. RESULTS: We use the median reconstruction at the synteny block level, of the ancestral genome of the order Gentianales, based on coffee, Rhazya stricta and grape, to exemplify the effects of resolution (granularity) on comparative genomic analyses. CONCLUSIONS: We show how decreased resolution blurs the differences between evolving genomes, with respect to rate, mutational process and other characteristics.

An engineered combinatorial module of transcription factors boosts production of monoterpenoid indole alkaloids in Catharanthus roseus.

To fend off microbial pathogens and herbivores, plants have evolved a wide range of defense strategies such as physical barriers, or the production of anti-digestive proteins or bioactive specialized metabolites. Accumulation of the latter compounds is often regulated by transcriptional activation of the biosynthesis pathway genes by the phytohormone jasmonate-isoleucine. Here, we used our recently developed flower petal transformation method in the medicinal plant Catharanthus roseus to shed light on the complex regulatory mechanisms steering the jasmonate-modulated biosynthesis of monoterpenoid indole alkaloids (MIAs), to which the anti-cancer compounds vinblastine and vincristine belong. By combinatorial overexpression of the transcriptional activators BIS1, ORCA3 and MYC2a, we provide an unprecedented insight into the modular transcriptional control of MIA biosynthesis. Furthermore, we show that the expression of an engineered de-repressed MYC2a triggers a tremendous reprogramming of the MIA pathway, finally leading to massively increased accumulation of at least 23 MIAs. The current study unveils an innovative approach for future metabolic engineering efforts for the production of valuable bioactive plant compounds in non-model plants.

Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis.

Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co-opted chemicals: the defence de-escalation hypothesis. We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA-adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de-escalation. We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited. Our findings are consistent with PA de-escalation as an adaptive response to specialist attack.

Unravelling the genetic differentiation among varieties of the Neotropical savanna tree Hancornia speciosa Gomes.

Background and Aims: Spatial distribution of species genetic diversity is often driven by geographical distance (isolation by distance) or environmental conditions (isolation by environment), especially under climate change scenarios such as Quaternary glaciations. Here, we used coalescent analyses coupled with ecological niche modelling (ENM), spatially explicit quantile regression analyses and the multiple matrix regression with randomization (MMRR) approach to unravel the patterns of genetic differentiation in the widely distributed Neotropical savanna tree, Hancornia speciosa (Apocynaceae). Due to its high morphological differentiation, the species was originally classified into six botanical varieties by Monachino, and has recently been recognized as only two varieties by Flora do Brasil 2020. Thus, H. speciosa is a good biological model for learning about evolution of phenotypic plasticity under genetic and ecological effects, and predicting their responses to changing environmental conditions. Methods: We sampled 28 populations (777 individuals) of Monachino's four varieties of H. speciosa and used seven microsatellite loci to genotype them. Key Results: Bayesian clustering showed five distinct genetic groups (K = 5) with high admixture among Monachino's varieties, mainly among populations in the central area of the species geographical range. Genetic differentiation among Monachino's varieties was lower than the genetic differentiation among populations within varieties, with higher within-population inbreeding. A high historical connectivity among populations of the central Cerrado shown by coalescent analyses may explain the high admixture among varieties. In addition, areas of higher climatic suitability also presented higher genetic diversity in such a way that the wide historical refugium across central Brazil might have promoted the long-term connectivity among populations. Yet, FST was significantly related to geographic distances, but not to environmental distances, and coalescent analyses and ENM predicted a demographical scenario of quasi-stability through time. Conclusions: Our findings show that demographical history and isolation by distance, but not isolation by environment, drove genetic differentiation of populations. Finally, the genetic clusters do not support the two recently recognized botanical varieties of H. speciosa, but partially support Monachino's classification at least for the four sampled varieties, similar to morphological variation.

Evolution on the backbone: Apocynaceae phylogenomics and new perspectives on growth forms, flowers, and fruits.

PREMISE OF THE STUDY: We provide the largest phylogenetic analyses to date of Apocynaceae in terms of taxa and molecular data as a framework for analyzing the evolution of vegetative and reproductive traits. METHODS: We produced maximum-likelihood phylogenies of Apocynaceae using 21 plastid loci sampled from 1045 species (nearly 25% of the family) and complete plastomes from 73 species. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Apocynaceae. KEY RESULTS: We obtained a well-supported phylogeny of Apocynaceae, resolving poorly understood tribal and subtribal relationships (e.g., among Amsonieae and Hunterieae, within Asclepiadeae), rejecting monophyly of Melodineae and Odontadenieae, and placing previously unsampled and enigmatic taxa (e.g., Pycnobotrya). We provide new insights into the evolution of Apocynaceae, including frequent shifts between herbaceousness and woodiness, reversibility of twining, integrated evolution of the corolla and gynostegium, and ancestral baccate fruits. CONCLUSIONS: Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are sensitive to choice of phylogenetic frameworks and models.

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases.

Biotechnology of the medicinal plant Rhazya stricta: a little investigated member of the Apocynaceae family.

Rhazya stricta Decne. (Apocynaceae) is an important medicinal plant that is widely distributed in the Middle East and Indian sub-continent. It produces a large number of terpenoid indole alkaloids (TIAs) some of which possess important pharmacological properties. However, the yields of these compounds are very low. Establishment of a reliable, reproducible and efficient transformation method and induction of hairy roots system is a vital prerequisite for application of biotechnology in order to improve secondary metabolite yields. In the present review, recent biotechnological attempts and advances in TIAs production through transformed hairy root cultures in R. stricta are reviewed to draw the attention to its metabolic engineering potential.

Analysis of transcriptional response to heat stress in Rhazya stricta.

BACKGROUND: Climate change is predicted to be a serious threat to agriculture due to the need for crops to be able to tolerate increased heat stress. Desert plants have already adapted to high levels of heat stress so they make excellent systems for identifying genes involved in thermotolerance. Rhazya stricta is an evergreen shrub that is native to extremely hot regions across Western and South Asia, making it an excellent system for examining plant responses to heat stress. Transcriptomes of apical and mature leaves of R. stricta were analyzed at different temperatures during several time points of the day to detect heat response mechanisms that might confer thermotolerance and protection of the plant photosynthetic apparatus. RESULTS: Biological pathways that were crosstalking during the day involved the biosynthesis of several heat stress-related compounds, including soluble sugars, polyols, secondary metabolites, phenolics and methionine. Highly downregulated leaf transcripts at the hottest time of the day (40-42.4 °C) included genes encoding cyclin, cytochrome p450/secologanin synthase and U-box containing proteins, while upregulated, abundant transcripts included genes encoding heat shock proteins (HSPs), chaperones, UDP-glycosyltransferase, aquaporins and protein transparent testa 12. The upregulation of transcripts encoding HSPs, chaperones and UDP-glucosyltransferase and downregulation of transcripts encoding U-box containing proteins likely contributed to thermotolerance in R. stricta leaf by correcting protein folding and preventing protein degradation. Transcription factors that may regulate expression of genes encoding HSPs and chaperones under heat stress included HSFA2 to 4, AP2-EREBP and WRKY27. CONCLUSION: This study contributed new insights into the regulatory mechanisms of thermotolerance in the wild plant species R. stricta, an arid land, perennial evergreen shrub common in the Arabian Peninsula and Indian subcontinent. Enzymes from several pathways are interacting in the biosynthesis of soluble sugars, polyols, secondary metabolites, phenolics and methionine and are the primary contributors to thermotolerance in this species.

Going west - A subtropical lineage (Vincetoxicum, Apocynaceae: Asclepiadoideae) expanding into Europe.

Vincetoxicum sensu lato is a tropical lineage comprising two clades that have reached high northern latitudes. Of the temperate clades, one is restricted to the Far East, the other one (Vincetoxicum s. str. Clade) extends into Europe, but their ranges overlap in Central China and Japan. Three species invasive in North America, V. hirundinaria, V. nigrum and V. rossicum, are members of the Vincetoxicum s. str. Clade. We explore the prerequisites for the range expansion in the Vincetoxicum s. str. Clade performing Bayesian and Maximum Likelihood phylogenetic analyses on sequences of the nuclear internal transcribed spacer (ITS) region, the nuclear external transcribed spacer region (ETS), and five plastid markers. The resulting phylogeny is used to conduct biogeographic analysis using BioGeoBEARS to reconstruct ancestral species ranges. Moreover, we map the known occurrences of two rare characters in Asclepiadoideae, the possession of phenanthroindolizidine alkaloids and reported cases of autogamy onto our phylogeny. Finally, we have conducted ecological niche modelling using Maxent on a total of 220 spatially unique occurrences of nine Vincetoxicum s. str. species spanning more than 4,000 km along the east-west gradient to learn about the climatic conditions along the presumed migration route. Our results indicate a north-westward migration in Vincetoxicum s. str. along the Asian mountain chains to Europe. Climatic preferences of the nine species sampled are dissimilar, except for the common exposure to at least one month of subfreezing temperatures, indicating a rather wide climatic tolerance for the clade as a whole. The three species invasive in North America belong to the northern Eurasian subclade and show the rare combination of phenanthroindolizidine alkaloids and autogamy.

Genetic diversity in natural populations of mangaba in Sergipe, the largest producer State in Brazil.

Mangaba (Hancornia speciosa Gomes) is found in areas of coastal tablelands in the Brazilian Northeast and Cerrado regions. This species has been subjected to habitat fragmentation that is mainly due to human activity, and requires conservation strategies. The aim of this study was to analyze the structure and inter- and intrapopulation genetic diversity of natural populations of H. speciosa Gomes using inter-simple sequence repeat (ISSR) molecular markers. A total of 155 individuals were sampled in 10 natural populations (ITA, PAC, IND, EST, BC, PIR, JAP, BG, NEO, and SANT) in the State of Sergipe, Brazil. Fifteen primers were used to generate 162 fragments with 100% polymorphism. Genetic analysis showed that the variability between populations (77%) was higher than within populations (23%). It was possible to identify five different groups by the unweighted pair group method with arithmetic mean and principal coordinate analysis, and only one individual (E10) remained isolated. Using ISSR markers it was possible to obtain a molecular profile of the populations evaluated, showing that these markers were effective and exhibited sufficient polymorphism to estimate the genetic variability of natural populations of H. speciosa Gomes.

A Set of 20 New SSR Markers Developed and Evaluated in Mandevilla Lindl.

Mandevilla is an ornamental crop with a bright future worldwide because of its high commercial acceptance and added value. However, as with most ornamental species, there are few molecular tools to support cultivar breeding and innovation. In this work, we report the development and analysis of 20 new Simple Sequence Repeat (SSR) markers in Mandevilla. Microsatellites were isolated from two enriched small-insert genomic libraries of Mandevilla × amabilis. The diversity parameters estimated after their amplification in a group of 11 commercial genotypes illustrate the effect of two opposite drifts: the high relatedness of cultivars belonging to the same commercial group and the high divergence of other cultivars, especially M. × amabilis. Based on their different band patterns, six genotypes were uniquely distinguished, and two groups of sport mutations remained undistinguishable. The amplification of the SSRs in three wild species suggested the existence of unexploited diversity available to be introgressed into the commercial pool. This is the first report of available microsatellites in Mandevilla. The development process has provided some clues concerning the genome structure of the species, and the SSRs obtained will help to create new products and to protect existing and upcoming plant innovations.

Recent radiation of Brachystelma and Ceropegia (Apocynaceae) across the Old World against a background of climatic change.

The genera Brachystelma Sims and Ceropegia L. of the Ceropegieae (Apocynaceae-Asclepiadoideae) consist of ±320 species of geophytes and slender climbers with a tendency to stem-succulence in Ceropegia. They occur in and around the semi-arid, mainly tropical parts of the Old World. For 146 species (around half of the total) from most of the geographic range of the genera, we analysed data from two nuclear and five plastid regions. The evolution of Ceropegia is very complex, with at least 13 mostly well-supported lineages, one of which is sister to the ±350 species of stapeliads. Species of Brachystelma have evolved at least four times, with most of them nested within two separate major lineages. So, neither Brachystelma nor Ceropegia is monophyletic. We recover a broad trend, in two separate major lineages, from slender climbers to small, geophytic herbs. Several clades are recovered in which all species possess an underground tuber. Small, erect, non-climbing, geophytic species of Ceropegia with a tuber are nested among species of Brachystelma. Consequently, the distinctive tubular flowers used to define Ceropegia do not reflect relationships. This re-iterates the great floral plasticity in the Ceropegieae, already established for the stapeliads. Both major lineages exhibit a trend from tubular flowers with faint, often fruity odours, pollinated by very small Dipteran flies, to flatter flowers often with a bad odour, pollinated by larger flies. Most of the diversity in Brachystelma and Ceropegia is recent and arose within the last 3my against a background of increased aridification or extreme climatic variability during the Pliocene. In the ingroup, diversity is highest in Southern Africa, followed by Tropical East Africa and other arid parts of Africa, the Arabian Peninsula and India. Many disjunctions are revealed and these are best explained by recent, long distance dispersal. In Africa, the diversity arises from the presence of many different lineages over wide areas but there is also evidence of closely related species growing together with different pollinators.

Development of microsatellite markers for Hancornia speciosa Gomes (Apocynaceae).

Herein, we describe 34 microsatellite loci developed using an enrichment genomic library for the tree species Hancornia speciosa Gomes (Apocynaceae). Thirty-five individuals were genotyped using 34 primers to analyze the polymorphisms at each locus. The number of alleles per locus ranged from 4 to 20. The average number of alleles was 8.11, and the expected heterozygosity ranged from 0.62 to 0.94. These microsatellite primers will be useful in population genetics studies for this species.